BME610 Shamik Sen

7/31/2019 BME610 Shamik Sen

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BME 610

Experimental & Quantitative Physiology

Prof. Shamik Sen

Contact Information:

Email: [email protected]

Office: 203, Civil Engg. Bldg.

Atomic Force Microscopy
mailto:[email protected]:[email protected]


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Extracellular Matrix (ECM)

Provides structural support to maintain tissue architecture (collagen, elastin)Provides mechanical strength to tissues (elastin)Acts as a reservoir of water molecules (proteoglycans)Supports cell adhesion (fibronectin, collagen, laminin)

Sequesters various chemical signals relevant to cell behavior


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Mechanical Properties of Cells

Stiffness /Youngs Modulus (E)

1 kPa 10 kPa 100 kPafluid

Brain MuscleBlood Collagenous Bone


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Mechanical Properties of Cells

Plasma Membrane

0.01- 0.1 mN/mfor most cellsTo

Membrane Tension (T0)

Contractility/Pre-stress (s)

Adhesion Strength/Adhesiveness


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Cell mechanical properties are altered in development & disease

Cell functioning is intimately tied to its physical properties

Cellular propertiesare dynamic in nature

Cellular propertiesevolve duringdevelopment

Alterations in cellphysical propertiesunderlie various diseases


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Biophysical techniques for probingcells & sub-cellular organelles

Apply Force to biological samples

Measure or record deformation of/in cell(s) cellular or sub-cellular level

Determine topography and/or physical propertyof cell(s) and/or sub-cellular compartments

Mechanical

Magnetic

Electrical

Optical

Force


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Atomic Force Microscope (AFM)

Invented in 1986 by Binnig & Rohrer at IBM, Zurich; commercialized in 1989

Offshoot of Scanning Tunneling Microscope (STM)

Can image non-conducting samples

Can image live samples; no requirement for fixing

Can be used for imaging as well as force measurements


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Comparison of biophysical tools


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A Typical AFM setup

Controller

Visualization Software

Inverted Microscope

AFM Head


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a sharp tip mounted on a flexiblecantilever that acts as a spring

a laser diode, whose light isfocused on the very end of thecantilever beam

a position-sensitive photodiode

that detects the laser beamreflected by the cantilever

a piezo device for positioning thesample relative to the tip

Principal Components of an AFM


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Cantilever & Tip Parameters


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Cantilever & Tip Parameters

Length (100-300 mm)

Width (10-50 mm)

Thickness (0.5 10 mm)

Shape (Rectangular, Straight, etc)

Material (Si, SiN)

Reflective coating (Al, Au)

Spring Constant (0.01-100 N/m)

Resonance Frequency (50-200 kHz)

Tip Radius (10-50 nm)

Tip Height (0-50 mm)

Tip Angle (15-infinity )

Tip Shape (3-sided, 4-sided)

Tip Material (Si, SiN, etc)


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Mode of Operation: Contact (DC) Mode

Tip & sample are in contact

Tip raster scans the surface

Sharp tip is used for contact mode

Deflection set point is specified

Used for very flat & rigid surfaces


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Mode of Operation: Tapping (AC) Mode

Tip & sample are not in perennial contact

Tip is oscillated at resonance frequency

Amplitude set-point is used Used for soft, loosely attached samples


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Mode of Operation: Force Mode

Tip & sample distance is varied


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Imaging Biological Samples: Effect of Gain


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Gain Control


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Low Gain Vs High Gain


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Setting Setpoint


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Setting Setpoint


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Setting Scan Rate


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Setting Scan Rate


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Gain, Setpoint, and Rate all greatly contributeto image quality and data fidelity.

There are no hard-and-fast rules or values for

any of these parameters.

Good image quality usually relies on aninterplay of different values for these parameters

In general, adjustments to values should startwith gain, then setpoint, and finish with rate.

Optimizing Gain, Setpoint & Rate


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Some Images with Optimized Parameters

DNA in Liquid E-Coli


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Osteoblast Cell

Endothelial Cell

Endothelial Cell(Mannitol treated)

h


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Force Measurements with AFM

What Can Force Curves Tell Us?

Adhesion: receptor-ligand bindingElasticity: cell stiffnessTethered eventProtein unfoldingBinding

Hookes Law:F = k * x = forcek= spring constant/stiffnessx= displacement of the spring

BUT First:

Calibrate the tip!!


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C d f i h dh i


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Force Curve: Hard surface with adhesion


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Cantilever Stiffness & Resonance Frequency

1/2

2

2

~ ( / )

0.5

0.5

/

c eff

B

c

c B

k m

E k T

E k d

k k T d

Non-destructiveEasy to do


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F C S ft f ith dh i


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Force Curve: Soft surface with adhesion

Di t d F C Fib bl t


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Directed Force Curves: Fibroblasts

E t ti Stiff V l


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Extracting Stiffness Values

2

2

2

tan 1

cellEF

3/ 2 1/ 2

2

4

3 1

cellEF R

Sneddons Model Hertz Model

Q tit ti El ti it M t


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Patterned cardiac myocyte on PDMS/glassCourtesy: Prof. K. Parker & Dr. N. Geisse,Harvard University

Quantitative Elasticity Measurements

Q tit ti El ti it M t


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Quantitative Elasticity Measurements

Topography/stiffness map of a lamellipodia of a spreading fibroblast(Solon et al., Biophys 07)

D t V A tif t i F M t


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Data Vs Artifact in Force Measurements

Do these varying values reflect heterogeneity of thecell or are these artifacts??

Data Vs Artifact in Force Measurements:


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Data Vs Artifact in Force Measurements:What are you probing?

Membrane Vs Cytoskeleton

A

B


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Choice of tip geometry in probing cells


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Choice of tip geometry in probing cells

Sharp tips may rupture cell membrane

Sharp tips may pass through pores in the cell membrane

Sharp tip provides very localized information of stiffness

Spherical tip more gentle on cells

Spherical tip provides averaged stiffness information

Effect of location of probing more important for sharp tips compared tospherical tips

St d i Adh i ith AFM


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Studying Adhesion with AFM

Receptor-ligand interactionsCell-cell interactions

Q tif i R d Bl d C ll M h


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Phagocyte

Signaling Self

Clustering

flow

RBC

Binding

CD47

SIRPa

Quantifying Red Blood Cell-MacrophageInteractions

Model System: Adhesively spread red cell


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Spread erythrocyte

glass (-)

poly-lysine layer (+++)

Model System: Adhesively spread red cell

negatively charged membrane constant cell volume constant membrane area

2.5 m

Imaged in AFM contact mode after glutaraldehyde fixation.

Strong Adhesion Limit Tensed Spherical Caps

AFM probing of unfixed cell membrane


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AFM probing of unfixed cell membrane

Indentation

z(nm)

Force(pN)

Indentation

Retraction

Adhesive detachment

0 200 400 600 800 1000

0

4000

8000

12000

z

Adhesion ?

Retraction with detachment

Probing CD47 SIRP interactions


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80

60

40

20

0

0 1 2 3 4

frequency

SIRP (human)

1

2

34

Human RBC

Rat RBC

# of peaks

Probing CD47- SIRP interactions

CD47 SIRP interaction is species restricted

Probing human CD47- human SIRP interactions


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0 100 200 300 400 500

0

500

1000

1500

2000

2500

Extension (nm)

Force(pN)

0 100 200 300 400 500

0

10

20

30

Peak to peak length (nm)

frequency

0 50 100 150 2000

10

20

30

Force (pN)

frequency

0 5 00 1 00 0 15 00 20 00 2 50 0 30 00

0

10

20

30

Force (pN)

frequency 0.5 mg/ml 0.05 mg/ml

2nd sawtooth1st sawtooth

SIRP SIRP

g

1500 pN first peak forces; 600 pN second peak forces at highconcentrations

At low concentration, peaks ~ 100 pN

215 nm mean extension

P k #1 Cl t #1

CD47-SIRP adhesion: Rate-dependence, & Valency of clusters


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Peak #2 = Cluster #2

Peak #1 = Cluster #1

~ 7 bonds break in parallel

< 4 bonds in parallel

Single bond strength:

70 pN @ 5 mm/sec

Second cluster shows:

fewer bonds

longer time needed

velocity ( m/s)

Indentation time (msec)

100

1000

0 5 10 15 20 25

e -time/500 msec

firstpkfo

rces(pN)

500 20 500 20

0 5 10 15 20 25

100

e -time/1200 msec

velocity ( m/s)

Indentation time (msec)

second

pkfo

rces(pN)

3 bonds break in parallel

~1 bond

CD47-SIRP Rate-dependence of Clustering -


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Phagocyte

Signaling Self

Clustering

flow

RBC

Binding

CD47

SIRPa

CD47-SIRP Rate-dependence of Clustering -implications for Signaling

tx ~ crossover from one cluster to two clusters

%

peaks

1 10

10

20

30

40

50

60

70

80

90

500 20Indentation time (msec)

Studying Force Spectroscopy with AFM


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Studying Force Spectroscopy with AFM

Protein Unfolding of Titin


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Protein Unfolding of Titin

Combined AFM & Microscopy


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Combined AFM & Microscopy

AFM: Provides high resolution images, and can characterize

biological forces and mechanics

Optical Microscopy: provides chemical/functional sensitivity; canlook below the surface of a specimen

Both: Can correlate fluorescently labeled structures withtopography

Mechanical stimulation with AFM


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Mechanical stimulation with AFM

BME610 Shamik Sen

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